The present invention relates to novel uses for 7xcex1-hydroxy-substituted steroids, to a process for preparing such steroids and to novel steroids so produced.
In particular the invention relates to the use of cytochromes of the cytochrome P450 family designated Cyp7b to effect 7xcex1-hydroxylation of certain 3xcex2-OH steroids so as to produce a 7xcex1-hydroxy-substituted steroids. Certain of the 7xcex1-hydroxy-substituted steroids so produced, as well the corresponding 7-oxo derivatives, are novel and form further aspects of the invention. The invention also relates to uses of these steroids, to uses of Cyp7b enzymes and to uses of novel macromolecular species, e.g. antibodies and DNAs, which are biologically related to the Cyp7b enzymes.
Cytochromes P450 are a diverse group of heme-containing mono-oxygenases (termed CYP""s; see Nelson et al., DNA Cell Biol. (1993) 12, 1-51) that catalyse a variety of oxidative conversions, notably of steroids but also of fatty acids and xenobiotics. While CYP""s are most abundantly expressed in the testis, ovary, placenta, adrenal and liver, it is becoming clear that the brain is a further site of CYP expression. Several CYP activities or mRNA""s have been reported in the nervous system but these are predominantly of types metabolizing fatty acids and xenobiotics (subclasses CYP2C, 2D, 2E and 4). However, primary rat brain-derived glial cells have the capacity to synthesize pregnenolone and progesterone in vitro. Mellon and Deschepper, Brain Res. (1993), 629, 283-292(9) provided molecular evidence for the presence, in brain, of key steroidogenic enzymes CYP11A1 (scc) and CYP11B1 (11xcex2) but failed to detect CYP17 (c17) or CYP11B2 (AS). Although CYP21A1 (c21) activity is reported to be present in brain, authentic CYP21A1 transcripts were not detected in this tissue.
Interest in steroid metabolism in brain has been fuelled by the finding that adrenal- and brain-derived steroids (neurosteroids) can modulate cognitive function and synaptic plasticity. For instance, pregnenolone and steroids derived from it are reported to have memory enhancing effects in mice. However, the full spectrum of steroid metabolizing CYP""s in brain and the biological roles of their metabolites in vivo has not been established.
Many aspects of brain function are modulated by steroids. Intracellular receptors for glucocorticoids (cortisol, corticosterone) are particularly abundantly expressed in the hippocampus (1), a brain region that plays a key role in specific aspects of memory formation, and which is an early and prominent target for dysfunction and damage in Alzheimer""s disease (AD). While glucocorticoids regulate learning and memory, mood and neuroendocrine control, chronic glucocorticoid excess compromises neuronal activity, synaptic plasticity and eventually survival, particularly in the hippocampus. These findings prompted the suggestion that glucocorticoid-mediated neurotoxicity might underpin some age-related brain disorders, including AD, in which plasma cortisol levels are markedly elevated (2).
Conversely, dehydroepiandrosterone (DHEA), the most abundant steroid product of the human adrenal cortex, has been proposed to protect against disorders of the aging brain (3). Plasma levels of DHEA often show a striking age-associated decline which correlates with loss of cognitive function (4). In rodents, injection of DHEA or its sulfate into limbic structures improves post-training memory and enhances synaptic plasticity (5). DHEA and glucocorticoids thereby appear to exert inverse effects upon memory function and synaptic plasticity, and DHEA has been advocated as an endogenous xe2x80x98anti-glucocorticoidxe2x80x99. However, despite considerable circumstantial evidence to support this contention, there is no evidence for a direct interaction between DHEA and glucocorticoid signalling pathways in neurons.
Neurosteroidogenesis has been reported in isolated rat retina (8) and brain (9). In addition to the production of pregnenolone and DHEA from cholesterol, a variety of novel steroids are made in brain extracts or cultured brain cells, including 20xcex1-dehydropregnenolone, 7xcex1-hydroxy derivatives of pregnenolone and DHEA, progesterone, and both 3xcex1- and 3xcex2-hydroxy-5xcex1-pregnan-20-one (reviewed in Ref. 7). Androgens are also modified, particularly through the action of aromatase and a 5xcex1-reductase (reviewed in Ref. 10). However, the specific enzymes responsible for these and other transformations in the central nervous system have not been well characterized.
As referred to above, several Cyps are present in the central nervous system (11-22). Activities or mRNAs corresponding to key steroidogenic enzymes (23-25), in addition to Cyp19 (aromatase) have been detected. Furthermore, mRNAs encoding the non-Cyp hydroxysteroid dehydrogenases (HSD) 3xcex1-HSD, 3xcex2-HSD and 11xcex2-HSD have been reported in the central nervous system (25, 27-29).
To investigate regulation of brain function, studies reported in copending International Patent Application No PCT/GB95/02465, published as WO 96/12810, and in Stapleton et al (J. Biol. Chem. 270, 29739-1995, Dec. 15, 1995), focused on the hippocampus, a brain region important in learning and memory. A copy of the specification of International Patent Application No PCT/GB95/02465 has been filed with the priority documents filed in respect of this specification.
That copending application, PCT/GB95/02465, describes and claims novel cytochrome P450 proteins designated Hct-1. These Hct-1 proteins have now been named as Cyp7b by the Committee on Standardized Cytochrome P450 Nomenclature and the name Cyp7b will be used in this application.
The Cyp7b enzyme shares 39% sequence identity to hepatic cholesterol 7xcex1-hydroxylase (Cyp7a) and lesser but significant homology with other steroidogenic Cyps. The postulated steroidogenic domain (30,31), found in many of these enzymes, is present in both Cyp7a and Cyp7b. Cyp7b mRNA is predominantly expressed in rodent brain, particularly in the hippocampus, unlike Cyp7a, which is liver-specific (31-33 and EP0648840 A2).
The present inventors have now investigated the substrate specificity of Cyp7b and found that Cyp7b catalyses the introduction of a hydroxyl group at the 7xcex1 position in steroid substrates, particularly 3xcex2-hydroxy steroids. Cytochromes Cyp7b are thus steroid hydroxylase enzymes having 7xcex1-specificity. The ability to produce 7xcex1-hydroxylated steroids is of major commercial importance, because such steroids are of particular use in the manufacture of pharmaceuticals (either as drugs per se or as intermediates), and in the manufacture of test kits and assays for pathological conditions associated with the presence of abnormal levels of endogenous enzyme, substrate or product.
The abbreviation xe2x80x9cDHEAxe2x80x9d will be used herein to designate dehydroepiandrosterone, thus 7xcex1-hydroxy-DHEA designates 7xcex1-hydroxydehydroepi-androsterone.
The present inventors have identified substrate/product pairs associated with Cyp7b, particularly DHEA/7xcex1-hydroxy-DHEA (7-HD), pregnenolone/7xcex1-hydroxy-pregnenolone (7-HP) and xcex2-estradiol/7xcex1-hydroxy-xcex2-estradiol (7-HE). They have also determined that DHEA concentration in brain tissue declines with age, whereas the concentrations of other brain steroids do not, and determined that the ageing process may be associated with deficits in certain steroids and also with deficits in the concentration of Cyp7b itself. It is also believed that one of the products produced by Cyp7b mediated reactions, namely 7xcex1-hydroxy dehydroepiandrosterone, plays an important role in the operation of the immune system. Because 7xcex1-hydroxy-DHEA is believed to be made substantially only in the brain, the inventors hypothesize that senescence may be due to a deficit in brain-produced 7xcex1-hydroxy-DHEA as well as in other steroids found in the brain such as DHEA, pregnenolone and 7xcex1-hydroxy-pregnenolone.
The present inventors have now further determined that one of the specific properties of the 7xcex1-hydroxy-substituted steroids, and potentially their 7-oxo substituted steroid derivatives, provided by the present invention is that of glucocorticoid and/or mineralocorticoid antagonism, whether at receptor level or otherwise. This is particularly demonstrated by the Example 5 below with respect to 7xcex1-hydroxy-DHEA but is more generally applicable. Thus this activity not only gives further uses for the novel steroids of the invention but provides first and second medical uses for known 7xcex1-hydroxy or 7-oxo steroids made available by the present process as glucocorticoid and/or mineralocorticoid antagonists and preferably in antagonism specific to neuronal tissue such as in the CNS.
Thus, having regard to this activity and their involvement in endogenous metabolic pathways, particularly in the brain, the 7xcex1-hydroxy substituted 3xcex2-hydroxy-steroids provided by use of the Cyp7b enzyme activity, including novel compounds provided by the invention, and their 7-oxo derivatives, have utility in the therapy of neuropsychiatric, immune and endocrine disorders, particularly but not exclusively steroid associated disorders.
Use of these 7xcex1-hydroxy or 7-oxo substituted 3xcex2-hydroxy-steroids, preferably possessing the carbon skeleton of cholesterol, androsterone, pregnenolone or estradiol, or derivatives thereof substituted independently at one or both of the 7- and 3-positions with an ester or ether group, in treating these disorders and for manufacturing medicaments for such treatment is provided in a first aspect of the present invention. Particularly preferred derivatives are those wherein one or both of the ester and or ether group is metabolisable in vivo to produce the corresponding hydroxy compound.
Preferred derivatives include those wherein the steroid has a 3xcex2-substituent-OR1 and/or a 7xcex1-substituent xe2x80x94OR2 where xe2x80x94OR1 and xe2x80x94OR2 each independently represents a free hydroxy, ester or ether group,
wherein each of R1 and R2 are independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-6 alkyl groups, groups R5COxe2x80x94, wherein R5 may be selected from substituted or unsubstituted C1-6 alkyl groups, and groups of the formula xe2x80x94OP(OH)3, wherein any substituents are selected from OH, halogen (F, Cl, Br, I) amino, C1-6 alkylamino, C1-6 dialkylamino, COOH or COOR4 wherein R4 represents a C1-6 alkyl group; and wherein the compounds may be in free form or in the form of acid addition salts with pharmacologically acceptable anions.
The particular disorders for which this utility is provided include
(a) deficits of cognition in aging
(b) Alzheimer""s disease
(c) deficits of immune system in aging
(d) deficits of immune function in HIV infection
(e) glucocorticoid or mineralocorticoid excess
(f) diabetes
(g) depression
(h) osteoporosis and hypercalcemia
(I) hyperglycemia and hyperlipodemia
(j) muscle atropy
(k) arterosclerosis
(l) steroid diabetes
Further, these 7xcex1-hydroxy steroids, their esters, ethers and 7-oxo derivatives may be used to induce cognitive enhancement in a normal individual.
Preferred steroids for such use have the carbon skeleton of androsterone, pregnenolone or estradiol and particularly preferred examples are 7xcex1-hydroxy-DHEA and 7xcex1-hydroxypregnenolone. Accordingly the present invention further provides the use of novel compounds of Formula Ia and Ib shown below in the applications indicated above.
Particularly preferred uses for the antagonistic properties of these 7-substituted steroids include treatment of disorders falling within category (e) above or where reversal of the effects of such corticoids, regardless of excess, is required.
A second aspect of the present invention provides pharmaceutical compositions implementing such use. The compositions in which the novel steroids and known steroids of the invention will be used will readily occur to those skilled in the art, generally comprising the steroid active in association with a pharmaceutically acceptable carrier or diluent, with formulations for example being suitable for inhalation or for gastrointestinal (e.g. oral), parenteral, topical, transdermal or transmucosal administration.
As an alternative to administering the compounds of the invention per se, a third aspect of the invention provides the possibility of using the gene sequences of the Cyp7b genes in gene therapy in order to compensate for a deficiency in Cyp7b enzyme. In such therapies, constructs comprising Cyp7b coding sequences can be packaged in conventional delivery systems, such as andenoviruses, vaccinia viruses, herpes viruses and liposomes and administered via a route which results in preferential targeting of a selected tissue, especially the brain. The invention further provides the possibility of using the gene sequences of the Cyp7b genes in gene therapy in order to achieve the endogenous expression of Cyp7b sequences for other purposes, e.g. in order to promote immunogenic processes. Thus for example, a vector such as a suitably modified vaccinia virus (or variant thereof) may be co-administered with a vaccine formulation so that the expressed Cyp7b sequences augment the immunogenic properties of the vaccine.
It will be realised that in the event of Cyp7b related disorders other than those involving its depletion it may be desirable to use vectors containing antisense sequences to Cyp7b effective such as to inhibit Cyp7b expression.
Macromolecules related immunologically to Cyp7b enzymes form fourth and fifth aspects of the invention and in this regard antibodies, particularly monoclonal antibodies which are capable of selectively binding Cyp7b, have utility in the diagnosis of disorders (a) to (l) referred to above. Anti-Cyp7b antibodies (including monoclonal antibodies) as well as binding molecules comprising antibody fragments may be produced by known methods and used in test kits for assays for Cyp7b enzymes.
According to a sixth aspect of the invention, there is provided a process of producing a 7xcex1-hydroxy-substituted steroid which comprises subjecting a corresponding steroid substrate having no hydroxyl substituent in the 7-position to hydroxylation in the presence of a Cyp7b steroid hydroxylase enzyme.
The Cyp7b steroid hydroxylase enzyme used in the process of the invention is preferably a Cyp7b enzyme described and claimed in the above-mentioned International Patent Application No PCT/GB95/02465 (and referred to therein as Hct-1). Such enzymes include (a) ones having the precise amino acid sequences described for mouse, rat and human Cyp7b, (b) homologous enzymes from other species and (c) enzymes having amino acid sequences which differ from the sequences of enzymes included in definitions (a) and (b), but in which the capacity to catalyse the introduction of a 7xcex1-hydroxyl group is not eliminated.
The amino acid sequence of suitable Cyp7b steroid hydroxylase enzymes may be defined in terms of the DNA coding sequences disclosed in International Patent Application No PCT/GB95/02465. Thus the Cyp7b steroid hydroxylase enzyme may have a sequence encoded by DNA coding sequences of Cyp7b enzymes selected from
(a) Coding sequences of DNA molecules comprising the coding sequence for rat Cyp7b set forth in SEQ Id No: 1,
(b) Coding sequences of DNA molecules comprising the coding sequence for mouse Cyp7b set forth in SEQ Id No: 2,
(c) Cyp7b steroid hydroxylase-encoding DNA molecules capable of hybridizing with the DNA molecule defined in (a) or (b) under standard hybridization conditions defined as 2xc3x97SSC at 65xc2x0 C.
20 (d) Cyp7b steroid hydroxylase-encoding DNA molecules capable of hybridizing with the DNA molecule defined in (a), (b) or (c) under reduced stringency hybridization conditions defined as 6xc3x97SSC at 55xc2x0 C.
The sequences (a) and (b) above represent rat and mouse Hct-1 gene sequence. Homologous sequences from other vertebrate species, especially mammalian species (including man) fall within the class of DNA molecules represented by (c) or (d).
Thus for human Cyp7b, the steroid hydroxylase enzyme may comprise a sequence encoded by
(e) DNA coding sequences selected from the following:
(i) the sequence designated xe2x80x9cexon 3xe2x80x9d in SEQ Id No 3,
(ii) the sequence designated xe2x80x9cexon 4xe2x80x9d in SEQ Id No 3, and
(f) Cyp7b steroid hydroxylase-encoding DNA molecules capable of hybridizing with the DNA molecules defined in (e) under standard hybridization conditions defined as 2xc3x97SSC at 65xc2x0 C.
(g) Cyp7b steroid hydroxylase encoding DNA molecules capable of hybridizing with the DNA molecule defined in (e) or (f) under reduced stringency hybridization conditions defined as 6xc3x97SSC at 55xc2x0 C.
(h) Cyp7b steroid hydroxylase-encoding DNA molecules comprising contiguous pairs of sequences selected from
(i) the sequence designated xe2x80x9cexon 3xe2x80x9d in SEQ Id No 3,
(ii) the sequence designated xe2x80x9cexon 4xe2x80x9d in SEQ Id No 3, and
(i) Cyp7b steroid hydroxylase-encoding DNA molecules capable of hybridizing with the DNA molecules defined in (h) under standard hybridization conditions defined as 2xc3x97SSC at 65xc2x0 C.
(j) Cyp7b steroid hydroxylase-encoding DNA molecules capable of hybridizing with the DNA molecule defined in (h) or (i) under reduced stringency hybridization conditions defined as 6xc3x97SSC at 55xc2x0 C.
(k) Coding sequences of DNA molecules comprising a contiguous coding sequence consisting of the sequences xe2x80x9cexon 3xe2x80x9d and xe2x80x9cexon 4xe2x80x9d in SEQ Id No 3, and
(l) Cyp7b steroid hydroxylase-encoding DNA molecules capable of hybridizing with the DNA molecules defined in (k) under standard hybridization conditions defined as 2xc3x97SSC at 65xc2x0 C.
(m) Cyp7b steroid hydroxylase-encoding DNA molecules capable of hybridizing with the DNA molecule defined in (k) or (l) under reduced stringency hybridization conditions defined as 6xc3x97SSC at 55xc2x0 C.
It will be appreciated that the DNA sequences referred to may consist of or be derived from genomic DNA, but typically would consist of or be derived from cDNA. Such sequences could be obtained by probing an appropriate library (cDNA or genomic) using hybridisation probes based upon the sequences provided according to the invention of International patent application No PCT/GB95/02465, or they could be prepared by chemical synthesis or by ligation of sub-sequences.
In the above definitions, Cyp7b steroid hydroxylases have been defined in terms of DNA sequence information. The Cyp7b steroid hydroxylase enzyme used in accordance with the process of the invention may alternatively or additionally be defined by reference to amino acid sequence information, e.g. the amino acid sequences contained in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO 6.
Thus the Cyp7b steroid hydroxylase enzyme used in accordance with the process of the invention may have sequences matching one of said sequences exactly, or alternatively, the enzymes used may have sequences which differ from the aforementioned sequences, provided that the capacity to catalyse the introduction of a 7xcex1-hydroxyl group is not eliminated.
Thus, for example, mutant enzymes may be produced by known methods, for example site-directed mutagenesis or other PCR-based procedures, and the expression products tested for their capacity to catalyse the introduction of a 7xcex1-hydroxyl group in selected substrates in accordance with the procedures described herein.
Having regard to the degree of homology between the rat, mouse and human enzymes and known data relating to species divergence of hydroxylase enzymes, it is preferred that by comparison with the DNA sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO.3, the mutant enzymes should should be encoded by sequences having at least 50% homology, more preferably at least 60% homology and most prefereably at least 70% homology with said sequences over a length of 50 contiguous nucleotides.
Preferably the mutant enzymes are encoded by sequences having at least 60% homology with the entire coding sequence, more preferably at least 70%.
Alternatively, by comparison with the amino acid sequences of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO.6, it is preferred that mutant enzymes should have at least 50% homology, more preferably at least 60% homology and most prefereably at least 70% homology with said sequences over a length of 30 contiguous amino acids. Preferably the mutant enzymes have at least 60% homology and more preferably 70% homology or more with the entire amino acid sequence in each case.
It is however preferred that such mutant enzymes do not differ too drastically from the aforementioned sequences and in this regard, where amino-acid substitutions are made, that the substituted amino acids are preferably so-called xe2x80x9csynonymousxe2x80x9d or xe2x80x9cconservativexe2x80x9d substitutions, i.e. hydrophilic, hydrophobic, basic and acidic amino acids should preferably be substituted by amino acids in the same class (see U.S. Pat. No. 5,380,712).
More specifically, it is preferred that the mutant enzymes differ from the precise sequences of those described herein by not more than 20, preferably not more than 10 and most preferably not more than 5 amino acid substitutions, insertions or deletions.
The Cyp7b enzymes described herein may be used in toxicological and drug evaluation studies and such uses form further aspects of the invention. In a particularly preferred embodiment of this aspect of the invention, a cell line capable of expressing a Cyp7b enzyme is used as a basis of an assay for one or more Cyp7b substrates. Such cell lines have utility in toxicological and drug evaluation studies. Most preferably the cell line comprises a prokaryotic or eucaryotic cell line which has been transformed so as artificially to express a Cyp7b enzyme. Examples include bacteria, yeast and mammalian cells. Also included are transgenic animals, at least one tissue of which (especially a non-brain tissue) expresses Cyp7b enzyme. Such transgenic animals may be produced by known methods for introducing foreign coding sequences into somatic or germ line cells.
The substrates used in the method of the invention are characterised by possessing a 3xcex2-hydroxyl group and further by preferably possessing the carbon skeleton of cholesterol, androsterone, pregnenolone or estradiol, with the proviso that where the substrate has the carbon skeleton of cholesterol, the substrate has a hydroxyl group in the 25, 26 or 27-position, preferably the 25-position.
Examples of such substrates include 25-hydroxycholesterol, dehydroepiandrosterone, pregnenolone and estradiol, in which case the steroids produced will be 7xcex1-hydroxy-25-hydroxycholesterol, 7xcex1-hydroxydehydroepiandrosterone, 7xcex1-hydroxy pregnenolone and 7xcex1-hydroxyestradiol (i.e. estra 1,3,5(10)-triene-3,7xcex1, 17xcex2-triol) respectively.
The 7xcex1-hydroxylated steroid produced according to the invention may be oxidised by known enzymatic or non-enzymatic procedures to produce 7-oxo substituted steroids and this further process step forms a further aspect of the invention.
Certain 7xcex1-hydroxy-substituted steroids produced according to the invention and certain corresponding 7-oxo derivatives are novel and provide a further aspect of the invention. Thus the present invention further provides novel 3xcex2-hydroxy steroids characterised in that they have a 7xcex1-hydroxy or 7-oxo substitutuent. Preferred novel steroids have the carbon skeleton of cholesterol, androsterone, pregnenolone or estradiol, with the provisio that where the skeleton is that of cholesterol, the 25, 26 or 27 position is hydroxylated, most preferably the 25 position.
Particular novel steroids are of the formula 
wherein OR1, OR2 and OR3 each independently represents a free hydroxy group, an ether group or an esterified hydroxy group.
In the case where OR1, OR2 and OR3 each independently represents an ether group, each of R1, R2 and R3 may be selected from substituted or unsubstituted C1-6 alkyl groups, any such substituents being selected from OH, halogen (F, Cl, Br, I) amino, C1-6 alkylamino, C1-6 dialkylamino, COOH or COOR4 wherein R4 represents a C1-6 alkyl group which may be unsubstituted or substituted by one of the substituents referred to above.
In the case where OR1, OR2 and OR3 each independently represents an esterified hydroxy group, each of R1, R2 and R3 may have the formula R5COxe2x80x94, wherein R5 may be selected from substituted or unsubstituted C1-60 alkyl groups, any such substituents being selected from OH, halogen (F, Cl, Br, I) amino, C1-6 alkylamino, C1-6 dialkylamino, COOH or COOR4 wherein R4 represents a C1-6 alkyl group; and groups of the formula xe2x80x94OP(OH)3. Where compounds of Formula Ia or Ib include substituents such as carboxyl groups, phospate groups, or substituted or unsubstituted amino groups, the compounds may be in free form or in the form of acid addition salts with pharmacologically acceptable anions (such as, for example, phosphate or halide ions) or cations (such as, for example, alkaline metal cations). Thus, where OR1, OR2 or OR3 represents hemesuccinate HOOC(CH2)2CO, the resulting hemesuccinate may be in the form of, for example, an Na or K salt.
It will be realised that the present invention provides for 7xcex1-hydroxylated and 7-oxo steroids as described above but which are further substituted at other positions directly on the steroid skeleton.
7xcex1-Hydroxyestradiol and 7-oxoestradiol are specific examples of compounds of Formula Ia and Ib.
The invention will now be described in more detail with particular reference to the following Figures and Examples.